Prolinimines: N-Amino- l -Pro-methyl Ester (Hydrazine) Schiff Bases from a Fish Gastrointestinal Tract-Derived Fungus, Trichoderma sp. CMB-F563

Article abstract of DOI:10.1021/acs.orglett.7b03666

A rice cultivation of a fish gastrointestinal tract-derived fungus, Trichoderma sp. CMB-F563, yielded natural products incorporating a rare hydrazine moiety, embedded within a Schiff base. Structures inclusive of absolute configurations were assigned to prolinimines A-D (1-4) on the basis of detailed spectroscopic and C₃ Marfey's analysis, as well as biosynthetic considerations, biomimetic total synthesis, and chemical transformations. Of note, monomeric 1 proved to be acid labile and, during isolation, underwent quantitative transformation to dimeric 3 and trimeric 4. Prolinimines are only the second reported natural products incorporating an N-amino-Pro residue, the first to include l-Pro, the first to occur as Schiff bases, and the first to be isolated from a microorganism.

Full text of DOI:10.1021/acs.orglett.7b03666

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Prolinimines: N‑Amino‑L‑Pro-methyl Ester (Hydrazine) Schiff Bases
from a Fish Gastrointestinal Tract-Derived Fungus, Trichoderma sp.
CMB-F563
Osama G. Mohamed, Zeinab G. Khalil, and Robert J. Capon*
Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia
S
* Supporting Information
ABSTRACT: A rice cultivation of a fish gastrointestinal tract-
derived fungus, Trichoderma sp. CMB-F563, yielded natural
products incorporating a rare hydrazine moiety, embedded within
a Schiff base. Structures inclusive of absolute configurations were
assigned to prolinimines A−D (1−4) on the basis of detailed
spectroscopic and C3 Marfey’s analysis, as well as biosynthetic
considerations, biomimetic total synthesis, and chemical trans-
formations. Of note, monomeric 1 proved to be acid labile and,
during isolation, underwent quantitative transformation to dimeric 3 and trimeric 4. Prolinimines are only the second reported
natural products incorporating an N-amino-Pro residue, the first to include L-Pro, the first to occur as Schiff bases, and the first to
be isolated from a microorganism.
n an effort to enhance investigations into the secondary
metabolites of Australian marine-derived fungi, we speculated
I
that commercial fish species, those with a feeding preference for
detritus and algae, may act as natural myco-accumulators. We
further hypothesized that such fish may be a readily accessible
source of fungal taxonomic, genetic, and chemical diversity. To
test these hypotheses, three Mugil mullet acquired from a local
fish market were used to assemble a library of ∼500
gastrointestinal tract-derived (GIT) fungal isolates. UPLC-
DAD profiling of the crude extracts obtained from agar plate
cultivations facilitated dereplication and revealed a remarkable
level of chemical productivity. This report provides an account of
our investigations into this fish GIT-derived resource, commenc-
ing with Trichoderma sp. CMB-F563.
Following cultivation trials on an array of media, UPLC-DAD
profiling revealed extracts obtained from rice grain cultivations of
Trichoderma sp. CMB-F563 as particularly rich in a diverse array
of secondary metabolites. To explore this potential, the EtOAc
extract obtained from a 45 d rice grain cultivation was subjected
to sequential solvent partitioning and trituration, followed by gel
and reversed-phase chromatography to yield known and new
phenolic bisanthraquinones, alkyl nucleosides, and aryl glyco-
sides (to be reported later).
More significantly, this fractionation also yielded an
unprecedented family of hydrazinyl Schiff bases, prolinimines
B−D (2−4, Figure 1), the structure elucidation of which was
secured by spectroscopic and C3 Marfey’s analysis, as well as total
biomimetic synthesis and chemical transformations, as summar-
ized below.
Figure 1. Prolinimines A−D (1−4).
axis). Further analysis identified a CO2CH3 moiety (δH 3.73,
CO2CH3; δC 52.7, CO2CH3; 175.8, CO2CH3) which 2D NMR
correlations (Figure 2) linked to an N-substituted proline methyl
ester residue, and after allowing for an axis of symmetry
accounted for 4 DBE. This conclusion was confirmed by a C3
Marfey’s analysis,1 which also established the L-Pro configuration
(Supporting Information). Consideration of NMR resonances
for the remaining elements of (C3HN)2, namely, two deshielded
HRESI(+)MS analysis of 2 afforded a sodium adduct ion
attributed to a molecular formula (C18H24N4O5, Δmmu −0.6)
requiring 9 double bond equivalents (DBE). The 1D NMR
(methanol-d4) data for 2 (Table 1) revealed symmetry (i.e., an
Received: November 26, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b03666
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. NMR (Methanol-d4) Data for Prolinimines A (1), B (2), and C (3)
1
2
3
Pos.
δH, m (J in Hz)
δC
δH, m (J in Hz)
δC
δH, m (J in Hz)
δC
furanyl F1
1
7.14, s
125.9
153.1
109.5
110.4
155.7
57.6
7.12, s
125.4
152.9
110.6
7.12, s
126.3
152.7
109.9
109.8
152.4
28.3
2
3
6.37, d (3.3)
6.32, d (3.3)
6.45, s
6.36, d (3.3)
6.15, d (3.3)
4
5
6
4.50, s
4.02, s
prolinyl P1
1′
2′
3′
175.8
66.1
29.4
175.8
66.1
29.5
175.9
66.1
29.4
4.28, dd (8.9, 3.1)
a 2.00, m
4.30, dd (8.9, 3.2)
a. 2.02, m
4.26, dd (8.9, 3.2)
a. 2.01, m
b 2.26, m
b. 2.27, m
b. 2.26, m
4′
5′
2.05, m
23.5
49.9
2.07, m
23.5
49.9
2.06, m
23.5
50.0
a 3.24, dd (16.9, 7.7)
b 3.46, m
a. 3.26, dd (16.9, 7.7)
b. 3.48, m
a. 3.23, dd (16.9, 7.7)
b. 3.46, m
CO2Me
3.72, s
52.7
3.73, s
52.7
3.72, s
52.7
prolinimine C (3) was assigned as shown, with the L-Pro absolute
configuration assigned on the basis of biosynthetic consid-
erations (cometabolite with 2) and total synthesis from L-Pro
(see below).
HRESI(+)MS analysis of 4 afforded a sodium adduct ion
attributed to a molecular formula (C35H42N6O9, Δmmu −2.2),
requiring 18 DBE. Although the quantity of 4 isolated from
CMB-F563 extracts did not allow for the acquisition of 1D and
2D NMR (DMSO-d6) data sufficient for unambiguous structure
assignment, the available data did nevertheless indicate the
presence of an asymmetric molecule containing three each of
furanyl, imine, and N-substituted-Pro methyl ester residues,
accounting for all DBE (Table 2). To solve the structure of 4
required total synthesis.
To confirm structures assigned to 2−3 and assign a structure
to 4, a convergent biomimetic synthesis was designed around the
putative biosynthetic precursors N-amino-L-Pro methyl ester
(5), 2,5-furandicarboxaldehyde (6), and 5-hydroxymethylfurfu-
ral (7) (Scheme 1). In a two-step process, L-Pro (8) was
converted to its N-nitroso methyl ester 9, which was in turn
reduced to 5.2,3 Exposure of 5 to 6 readily yielded the bis-Schiff
base prolinimine B (2), whereas exposure of 5 to 7 yielded the
mono-Schiff base prolinimine A (1) whose structure, including
imine configuration, was assigned by detailed spectroscopic
analysis (Figure 2) and comparisons to 2−4. Significantly,
exposure of 1 to slightly acidic conditions yielded 3, with
accompanying yields of prolinimine D (4). In summary,
prolinimines A−D (1−4) were prepared in nonoptimized yields
of 36, 50, 3, and 1%, respectively, from 8.
A plausible mechanism for the acid-mediated transformation
of 1 to 3−4 involves dehydration to form an activated furanyl
oxonium species, primed for dimerization and the loss of
formaldehyde to yield 3 and trimerization to 4 (Scheme 2). By
contrast, as 2 lacks an acid sensitive allylic 1°-OH moiety, it is
incapable of generating a furanyl oxonium and is inert to acid-
mediated transformation. Although acid modifiers were not used
during the isolation of prolinimines, fractions were nevertheless
acidic due to high concentrations of accompanying phenolic
bisanthraquinone cometabolites.
Figure 2. Diagnostic NMR correlations for 1−4.
sp2 methines (δH 7.12, δC 125.4; δH 6.45, δC 110.6) and one sp2
quaternary carbon (δC 152.9) as well as 5 unattributed DBE and
additional 2D NMR correlations (Figure 2), supported the
structure for prolinimine B (2) as indicated. The E imine
configuration was assigned on the basis of diagnostic ROESY
correlations between H-1 and H2-5′ (Figure 2).
HRESI(+)MS analysis of 3 afforded a sodium adduct ion
attributed to a molecular formula (C23H28N4O6, Δmmu −0.4),
requiring 12 DBE. As with 2, the 1D NMR (methanol-d4) data
for 3 (Table 1) indicated symmetry (i.e., an axis), with selected
resonances and correlations (Figure 2) readily attributed to N-
substituted Pro methyl ester residues linked through an imine to
a bis-furanyl moiety. Identical chemical shifts (δH 7.12) for H-1 in
3 and 2 supported a common E imine configuration, further
confirmed by diagnostic ROESY correlations between H-1 and
H2-5′. The 2,5-disubstituted bis-furanyl moiety in 3 was
evidenced by diagnostic 2D NMR correlations to and from C-
6/H2-6 at the axis of symmetry (Figure 2). Thus, the structure for
Significantly, synthetic samples of 2−4 were chemically
(UPLC-DAD) and spectroscopically (1D and 2D NMR,
HRESIMS, UV−vis, [α]D) identical to those isolated from
B
DOI: 10.1021/acs.orglett.7b03666
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 2. NMR (DMSO-d6) Data for Prolinimine D (4)
Pos.
δH, m (J Hz)
δC
Pos.
δH, m (J Hz)
δC
Pos.
δH, m (J Hz)
3.87, br s
δC
furanyl F1
furanyl F2
furanyl F3
1
7.06, s
123.6
150.9
108.2
108.7
150.4
26.9
7
-
150.0
110.4
118.8
146.3
123.2
12
23.4
153.1
107.8
2
8
6.07, s
13
-
A
3
6.34, d (3.3)
6.19, d (3.3)
9
-
14
6.07, d (3.3)
6.31, d (3.3)
-
A
4
10
11
-
15
108.2
5
7.10, s
16
150.6
123.9
6
4.00, s
17
7.05, s
prolinyl P1
prolinyl P2
prolinyl P3
B
B
E
B
1′
2′
3′
173.3
64.3
27.8
-
1″
2″
3″
173.3
64.5
27.7
-
1‴
2‴
3‴
173.3
A
C
D
A
A
C
4.22, dd (8.9, 2.3)
4.22, dd (8.9, 2.3)
4.22, dd (8.9, 2.3)
64.3
B
B
B
D
a. 1.91, m
a. 1.91, m
a. 1.91, m
27.8
C
C
C
b. 2.18, m
b. 2.18, m
b. 2.18, m
-
D
E
F
D
D
E
4′
5′
1.97, m
22.0
48.6
-
4″
5″
1.97, m
22.0
48.5
-
4‴
5‴
1.97, m
22.0
E
E
E
F
a. 3.14, m
a. 3.14, m
a. 3.14, m
48.6
F
F
F
b. 3.36, m
b. 3.36, m
b. 3.36, m
-
G
G
G
G
CO2Me
3.63, s
51.7
CO2Me
3.62, s
51.8
CO2Me
3.63, s
51.7
A−G
δH and δC assignments with the same superscript may be interchanged.
CMB-F563. With synthetic 4 in hand, we acquired higher quality
NMR (DMSO-d6) data (Supporting Information), revealing
diagnostic 2D NMR correlations, and in particular ROESY
correlations from H-11 to H2-12 (Figure 2), which established
the C-9 to C-12 substitution and provided an unambiguous
structure assignment for prolinimine D (4).
Scheme 1. Synthesis of 1−4
HPLCMS analyses of crude EtOAc extracts derived from
CMB-F563 rice cultivations successfully detected 1 and 2, but
not 3 nor 4. From these observations, we conclude that 1 is a
natural product and cometabolite of 2, albeit one that is acid
labile and in need of careful handling during extraction and
fractionation. Furthermore, although isolated from the crude
EtOAc extract, 3 and 4 are in fact acid-mediated handling
artifacts. Of note, the acid-mediated biomimetic transformation
of 1 to 3 and 4 reveals a promising process for regio-controlled
Scheme 2. Plausible Mechanism for the Acid-Mediated Interconversion of 1 to 3 and 4
C
DOI: 10.1021/acs.orglett.7b03666
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Organic Letters
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(4) Klaiklay, S.; Rukachaisirikul, V.; Phongpaichit, S.; Buatong, J.;
Preedanon, S.; Sakayaroj, J. Nat. Prod. Res. 2013, 27 (19), 1722.
(5) Li, H.; Saravanamurugan, S.; Yang, S.; Riisager, A. ACS Sustainable
Chem. Eng. 2015, 3 (12), 3274.
(6) Le Goff, G.; Ouazzani, J. Bioorg. Med. Chem. 2014, 22 (23), 6529.
(7) Mayengbam, S.; Yang, H.; Barthet, V.; Aliani, M.; House, J. D. J.
Agric. Food Chem. 2014, 62 (2), 419.
assembly of methylene interrupted 2,5-disubstituted furans.
While such furans are exceptionally rare among natural products,
being limited to flavodonfuran isolated from the mangrove
endophytic fungus Flavodon flavus PSU-MA201,4 they are of
considerable interest in the catalytic transformation of biomass-
derived carbohydrates to biodiesel.5
Naturally occurring hydrazines are also extremely rare, with
only a handful of nonacylated examples reported to date.6 A
particularly interesting and relevant example of an acylated
hydrazine (i.e., a hydrazide) is the flaxseed toxin linatine, which is
an amide conjugate of glutamic acid and the hydrazine N-amino-
D-Pro.2 In chickens reared on linseed meal (i.e., flaxseed) linatine
undergoes in vivo proteolysis to N-amino-D-Pro, which then
forms a Schiff base with and sequesters the aldehyde pyridoxine,
leading to vitamin B6 deficiency syndrome,7 rendering linseed
meal unsuitable as animal feed.
The fungal prolinimines represent only the second reported
occurrence of a naturally occurring metabolite incorporating an
N-amino-Pro residue. As many fungi are known to infect stored
grains, should any such strains produce hydrazine-based vitamin
B6 antagonists (i.e., N-amino-Pro), or closely related pro-drugs
(i.e., hydrazides or Schiff bases), they could render grain supplies
toxic. Forewarned is forearmed, and if recognized early, such
hydrazine intoxication could be treated with vitamin supple-
ments.7 Our discovery of the fungal origin, structures, and
chemistry of the hydrazinyl Schiff base prolinimines represents a
timely opportunity to raise awareness of the potential
contamination of foodstuffs by fungi-derived N-amino-L-Pro
(or related prodrugs).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b03666.
General experimental procedures, microbial taxonomy
and cultivation, purification, characterization, synthesis,
chemical interconversion, and spectroscopic data (tabu-
lated and spectra) for 1−4 (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: r.capon@uq.edu.au. Tel: +61-7-3346-2979. Fax: +61-7-
3346-2090.
ORCID
Robert J. Capon: 0000-0002-8341-7754
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank P. Abdala for assistance in fungal isolations.
O.G.M. acknowledges the provision of a University of Queens-
land, UQ International Scholarship. This work was funded in
part by the Institute for Molecular Bioscience and the University
of Queensland.
REFERENCES
■
(1) Vijayasarathy, S.; Prasad, P.; Fremlin, L. J.; Ratnayake, R.; Salim, A.
A.; Khalil, Z.; Capon, R. J. J. Nat. Prod. 2016, 79 (2), 421.
(2) Klosterman, H. J.; Lamoureux, G. L.; Parsons, J. L. Biochemistry
1967, 6 (1), 170.
(3) Waibel, M.; Hasserodt, J. J. Org. Chem. 2008, 73 (16), 6119.
D
DOI: 10.1021/acs.orglett.7b03666
Org. Lett. XXXX, XXX, XXX−XXX